Unveiling and initial characterization of neural crest-like cells in mesenchymal populations from the human periodontal ligament.

OBJECTIVE: To identify cells with neural crest properties in mesenchymal populations isolated from human periodontal ligament. BACKGROUND: Evidence from tracing experiments on animal embryos revealed proof that dental tissues are among the homing sites of craniofacial neural crest migratory cells. In humans, similar migratory cells were found in early embryos, but whether these cells are progeny of oral multipotent stem cells needs to be confirmed. Searching for the cells with neural crest characteristics in periodontal ligament mesenchymal populations can lead to a solution to the problem. MATERIAL AND METHODS: Populations from the human periodontal ligament were cultured in media supplemented with various concentrations of fetal bovine serum (FBS); assays were performed to evaluate the expression of neural crest, mesenchymal and multipotency genes. RESULTS: In periodontal ligament populations cultured in the standard expansion medium containing minimal amounts of FBS (0.5% or 1%) or lacking FBS, growing numbers of epithelial-like cells emerged, co-expressing neural crest-specific (p75, HNK-1, SOX10), the epithelialization (E-cadherin) and mesenchymal (CD73 and CD105) markers. CONCLUSION: The human periodontal ligament contains a subpopulation of dormant neural crest-like cells, which can be highlighted by culturing at FBS concentrations below 2% or in a serum-free medium.

Human dental pulp stem cells respond to cues from the rat retina and differentiate to express the retinal neuronal marker rhodopsin.

Human adult dental pulp stem cells (DPSCs) are self-renewing stem cells that originate from the neural crest during development and remain within the dental pulp niche through adulthood. Due to their multi-lineage differentiation potential and their relative ease of access they represent an exciting alternative for autologous stem cell-based therapies in neurodegenerative diseases. In animal models, DPSCs transplanted into the brain differentiate into functional neurons or astrocytes in response to local environmental cues that appear to influence the fate of the surviving cells. Here we tested the hypothesis that DPSCs might be able to respond to factors present in the retina enabling the regenerative potential of these cells. We evaluated the response of DPSCs to conditioned media from organotypic explants from control and chemically damaged rat retinas. To evaluate cell differentiation, we analyzed the expression of glial fibrillary acidic protein (GFAP), early neuronal and retinal markers (polysialic acid-neural cell adhesion molecule (PSA-NCAM); Pax6; Ascl1; NeuroD1) and the late photoreceptor marker rhodopsin, by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). Exposure of DPSC cultures to conditioned media from control retinas induced a 39% reduction on the number of DPSCs that expressed GFAP; the expression of Pax6, Ascl1, PSA-NCAM or NeuroD1 was undetectable or did not change significantly. Expression of rhodopsin was not detectable in control or after exposure of the cultures with retinal conditioned media. By contrast, 44% of DPSCs exposed to conditioned media from damaged retinas were immunopositive to this protein. This response could not be reproduced when conditioned media from Müller-enriched primary cultures was used. Finally, quantitative RT-PCR was performed to compare the relative expression of glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in DPSC co-cultured with retinal organotypic explants, where BDNF mRNA expression was significantly upregulated in retinal-exposed cultures. Our data demonstrate that DPSC cultures respond to cues from the rat retina and differentiate to express retinal neuronal markers.

Lesion sites that predict the ability to gesture how an object is used.

We used a two stage procedure to predict which stroke patients would have chronic difficulties gesturing how to use an object when object recognition and hand movements were intact. First, we searched our PLORAS database by behavior and identified 5 patients who had chronic difficulty gesturing object use but no difficulty recognising objects, comprehending words or moving their hands. High definition lesion analyses showed that all 5 patients had damage to the white matter underlying the left ventral supramarginal gyrus, (A) close to the cortex, (B) deep towards the midline and (C) extending into the temporal lobe. In addition, 2 patients had damage to (D) the left posterior middle temporal cortex, and 3 patients had damage to (E) the left dorsal supramarginal gyrus and (F) the left premotor cortex. Second, we searched our database by lesion location for patients who had damage to any part of regions ABCDEF. The incidence of gesturing difficulties was higher in patients with damage to ABCD (7/9), ABCE (7/10) or ABCDE (10/13) than ABCF (7/13), ABC (8/16) or partial damage to ABCF (6/32). Thus behaviour was best predicted by the combination of regions that were damaged (a "network-lesion") rather than on the basis of each region alone or overall lesion size. Our results identify which parts of the temporal and parietal lobes impair the ability to gesture object use and which parts need to be intact to support it after damage. Our methods provide a framework for future studies aiming to predict the consequences of brain damage.

Induction of immunity in sheep to Fasciola hepatica with mimotopes of cathepsin L selected from a phage display library.

An M13 phage random 12-mers peptide library was used to screen cathepsin L mimotopes of Fasciola hepatica and to evaluate their immunogenicity in sheep. Seven clones showed positive reactivity to a rabbit anti-cathepsin L1/L2 antiserum in ELISA, and their amino acid sequences deduced by DNA sequencing were tentatively mapped on the protein. Twenty sheep were randomly allocated into 4 groups of 5 animals each, for immunization with 1x10(14) phage particles of clones 1, 20, a mixture of 7 clones and PBS, without adjuvant at the beginning, and 4 weeks later. All groups were challenged with 300 metacercariae at week 6 and slaughtered 16 weeks later. The mean worm burdens after challenge were reduced by 47.61% and 33.91% in sheep vaccinated with clones 1 and 20, respectively; no effect was observed in animals inoculated with the clone mixture. Also, a significant reduction in worm size and burden was observed for those sheep immunized with clone 1. Animals receiving clone 20, showed a significant reduction in egg output. Immunization induced a reduction of egg viability ranging from 58.92 to 82.11%. Furthermore, vaccinated animals produced clone-specific antibodies which were boosted after challenge with metacercariae of F. hepatica.

L-Homoserine: a novel excreted metabolic marker of hepatitis B abnormally produced in liver from methionine.

The hepatitis B infection leads to various profound pathological processes in liver metabolism. Some biochemical alterations detectable by blood analysis are currently used for a preliminary evaluation of the infection. Based on existing data we present here evidence that non-protein amino acid L-homoserine is a pathological, hepatitis B-induced metabolite that is formed and excreted into urine from methionine via splitting S-adenosylmethionine. The urine L-homoserine is proposed as a new marker in the pre-diagnosis examinations that is easier for the clinical analysis than currently used blood test, and is applicable to large-scale epidemiological surveys of the probability of hepatitis B.

Post-panning computer-aided analysis of phagotope collections selected with neurocysticercosis patient polyclonal antibodies: separation of disease-relevant and irrelevant peptide sequences.

The homology of peptide sequences selected from a 7mer phage display library with antibodies elicited by the multicelled parasite Taenia solium in cerebrospinal fluid and serum of neurocysticercosis (NCC) patients and by antibodies of uninfected control patients with similar neurological complications of other ethiology (non-NCC) were analyzed using a PILEUP-Tudos sequence alignments program. The analysis generated dendrograms bearing two types of sequence clusters, those containing (1) only NCC patients-derived peptides and (2) both NCC- and control non-CC -- patient derivatives. By using ELISA, peptides that were selected by the antibodies were identified predominantly in the NCC-derived clusters. In repeated analysis in which sequences were added or removed, the first type of clusters maintained their structure, while the second type of clusters were split into many separate homology units dispersed throughout the guide tree. These results are interpreted as the ability of the analysis to segregate NCC-specific peptide sequences from other sequences. Altogether, this study demonstrates the high potential of the PILEUP-Tudos computer program to analyze phagotope collections recovered through biopanning with polyclonal antibodies elicited in patients by complex and as yet unknown multiple pathogenic antigens and to separate all phagotopes that are disease-relevant on the basis of the sequence homology.

Epitope mapping on N-terminal region of taenia solium paramyosin.

Epitope mapping of the amino-terminal 20aa sequence from Taenia solium paramyosin (TPmy), an immunodominant protein involved in the complex host-parasite relationship in human and porcine cysticercosis is reported. A 12-mer random peptide phage display library was screened with antibodies raised against a synthetic peptide corresponding to the amino-terminal 20aa sequence of TPmy, its highly immunodominant region. In total, 57 clones isolated in two panning conditions were analyzed, of which a single group of 14 sequences found in 25 clones shared a consensus motif showing structural similarity with the antigen Arg10-Thr16 region.

Isolation and structure-functional characterization of phage display library-derived mimotopes of noxiustoxin, a neurotoxin of the scorpion Centruroides noxius Hoffmann.

Noxiustoxin (NTX) is a short-chain toxin from the venom of the scorpion Centruroides noxius Hoffmann, whose molecular structure and physiological effects have been characterized in detail, whereas the antigenic properties of this and other K(+) channel-blocking toxins are poorly studied. A monoclonal antibody against NTX, BNTX18, able to inhibit the binding of NTX to rat brain synaptosomes, was used in the present study for selecting immunoreactive peptides, mimotopes, from a 12mer and a 7mer phage library. The peptides were characterized immunologically and used for mapping the epitope on NTX. In total, 75 phage clones carrying 43 different peptides were analyzed of which 42 clones carrying 17 different peptides, twelve 12mer and five 7mer peptides, presented a single consensus motif: Leu(Ile, Val)-Tyr(Phe, Trp, Leu)-Gly-Met(Ala). All but three of the peptides containing this motif were reactive with selected mAb BNTX18 in a dot-blot assay of which eight were clearly positive in ELISA and exhibited in competition-inhibition assay the antibody binding specificity of the NTX epitope recognized by BNTX18. The two most reactive mimotopes injected into mice showed the ability to induce antibodies reacting with NTX, thus, to mimic the epitope of NTX antigenically. Sequence comparison and the analysis of the three-dimensional structure of NTX led to the proposal that residues Glu19-Leu20-Tyr21-Gly22 and the hydrophobic part of the side chain of Lys18 form the C-terminal part of the epitope. Due to the frequent presence of residues Pro, Leu, Thr, Arg, and Gln in the N-terminal part of the mimotopes, corresponding homologous residues in the N-terminal proximity of the partial epitope may be part of an additional more hydrophilic epitope element.

[Targeting the gene for platelet-derived growth factor A-chain (PDGF-A) and preparation of chimeric mice with a "knockout" genome]. / Targeting gena A-tsepi trombotsitarnogo faktora rosta (PDGF-A) i poluchenie khimernoi myshi s "nokautirovannym" genom.

A vector was selected for homologous recombination to generate mouse embryonic stem cell clones with disrupted platelet-derived growth factor A (PDGF-A) chain gene. A chimeric mouse with targeted PDGF-A gene has been created.

[Determination of homoserine kinase activity by chromatographic separation and measurement of reaction products]. / Opredelenie aktivnosti gomoserinkinazy putem khromatograficheskogo razdeleniia i izmereniia produktov reaktsii.

A highly specific procedure for quantitative assay of the homoserine kinase activity in an optimized enzymatic reaction using 14C-labelled homoserine or [gamma 33P]-ATP as substrates, and paper or thin-layer chromatography for separation of the formed o-phosphohomoserine, is described. The procedure is simple, sensitive and allows the assay for the activity of both purified and non-purified homoserine kinases.

[Cloning and structural characteristics of human hair keratin genes rich in sulfur]. / Klonirovanie i strukturnaia kharakteristika genov bogatykh seroi keratinov volos cheloveka.

Two human genomic genes for the hair high-sulphur keratins were for the first time cloned in a 15 kb fragment. The primary structures of the coding regions of the genes and their 5'- and 3'-flanks were determined. In the 5'-flanking region, TATA boxes, initiating codons and a 18 nucleotide sequence, previously described in sheep keratin genes and designated as "the matrix-specific" sequence was revealed. Basing on the nucleotide sequences, the encoded amino acid sequences of the high-sulphur keratins were determined for the first time. The suggested functional role of the structural elements (regions) revealed in the proteins primary structure and problems concerning their evolution tendencies are discussed.

[Influence of the expression of the human growth hormone releasing factor gene on hormone levels in a transgenic rabbit]. / Vliianie ékspressii gena rilizing-faktora gormona rosta cheloveka na uroven' gormonov u transgennogo krolika.

A transgene rabbit with the human growth hormone releasing factor gene was produced by a method of microinjections into fertilized oocytes; a mouse metallothionein I gene was used as a promoter. Gene expression was accompanied by a phenotypical effect, expressed in increasing the rates of development. The maximum difference among the transformant, transplants and control was revealed on the 30-45th day of postnatal development. Analysis of the hormonal status of the transgene animal has shown change in the levels of the majority of hormones: a 6-10-fold increase in insulin; a 2-3-fold increase in the level of triiodothyronine, thyroxin; a reduced somatostatin concentration, a two-fold decrease in the level of progesterone, and a four-fold decrease in the level of testosterone. Activation of the promoter zone with Zn++ salts for 5 weeks resulted in a further increase in the transformant body mass by 10%. However blood hormone levels in the transgene rabbit returned to normal. Proceeding from the above it can be assumed that exogenous gene expression probably increased somatotropin secretion which determined dysfunction of most of the endocrine glands; the effect of somatotropin was probably insulin-mediated.

[Biological effect of human erythropoietin in transgenic mice]. / Biologicheskii éffekt éritropoétina cheloveka u transgennykh myshei.

Transgenic mice were obtained inheriting the human erythropoietin gene under the control of viral regulatory elements. The reliable difference in haematocrit, the content of haemoglobin and percentage of reticulocytes in peripheral blood were not revealed. The level of serum erythropoietin in transgenic mice is several fold higher than in control mice. The increased pool of erythroid cells was observed in the bone marrow of transgenic mice, especially of normoblasts (3-fold) and reticulocytes (4,5-fold).

[Localization of fragments binding nuclear proteins in the promotor region of the human alpha-1-antitrypsin gene]. / Lokalizatsiia v promotornoi oblasti gena alpha-1-antitripsina cheloveka uchastkov sviazyvaniia s iadernymi belkami.

Human and mice nuclear extracts from livers and mice spleen extract were analysed in an attempt to find any proteins capable of binding to the human alpha 1-antitrypsin gene promoter. The nuclei of all studied tissues contain such proteins. The proteins were partially purified on DEAE-trisacryl, heparin sepharose and phosphocellulose columns. The multiple sites for liver nuclear proteins binding to the human alpha 1-antitrypsin gene promoter were found by the DNAse I footprinting technique.

[Induction of unstable mutations in Drosophila melanogaster by microinjection of oncogenic virus DNA into polar embryonic plasm. Malignant effect of oncoviral DNA]. / Induktsiia nestabil'nykh mutatsii u Drosophila melanogaster mikroin''ektsei DNK onkogennykh virusov v poliarnuiu plazmu émbrionov. Maligniziruiushchii éffekt onkovirusnykh DNK.

We have demonstrated that the ability to induce benign neoplasms We have dominant mode of inheritance in Drosophila melanogaster is the specific feature of oncoviral DNAs. It is supposed that development of this type of neoplasms in Drosophila is connected with the changes in expression of protooncogenes in mutant genome: firstly, the genetic factors directing the development of neoplasms and Drosophila protooncogenes which shared the homology with v-src are localised in the same regions; secondly, there are structural rearrangements in c-src/fps (29A) protooncogene in mutant stocks which display the ability for neoplastic growth.

[Induction of unstable mutations in Drosophila melanogaster by microinjection of oncogenic virus DNA into the embryo polar plasma. Insertional nature of mutations]. / Induktsiia nestabil'nyukh mutatsii u Drosophila melanogaster mikroin"ektsiei DNK onkogennykh virusov v poliarnuiu plazmu émbrionov. Insertsionnaia priroda mutatsii.

We have demonstrated that mutations induced in Drosophila melanogaster by the microinjections of adenovirus Sa7 DNA in early embryos are of insertional nature. The role of insertional elements is played by the Drosophila transposons, but not by the virus DNA. The ability of oncoviral DNA to induce transpositions of mobile elements in recipient genome is the molecular basis of this system of genetic instability.

[Induction of unstable mutations in Drosophila melanogaster by microinjections of oncogenic virus DNA into the embryo polar plasma. Prolonged genetic instability of mutations at the lobe locus]. / Induktsiia nestabil'nykh mutatsii u Drosophila melanogaster mikroin"ektsiei DNK onkogennykh virusov v poliarnuiu plazmu émbrionov. Prodlennaia genet'icheskaia nestabil'nost' mutatsitii v lokuse lobe.

Mutations Lobe induced by the microinjections of RSV cDNA into Drosophila melanogaster early embryos are characterized by permanent genetic instability; the level of this instability is being changed in time. Based on the results of genetic analyses of Lobe mutations and molecular analysis of white and ADH mutations induced at high frequency in this system of gene instability, we supposed that unstable mutations which arose under the influence of retroviral cDNA are of the insertional nature.

[Design of a gene coding encoding a polypeptide, mixing the enzyme activity of Escherichia coli homoserine kinase and threonine synthetase]. / Konstruirovanie gena, kodiruiushchego polipeptid, sovmeshchaiushchii fermentativnye aktivnosti gomoserinkinazy i treoninsintetaty Escherichia coli.

The functioning of Escherichia coli threonine operon isolated genes thrB and thrC was studied by using the genetic complementation and enzymatic activity determination techniques. A new gene thrBC was obtained by the genes merging. The genes thrB and thrC were shown to function in Escherichia coli cells independent of the operon and the polipeptide encoded by the thrBC gene combined the functions to express the products of both genes in bacterial cell. At the same time the enzyme coded for by the merged genes demonstrates the level of activity compared with the ones of the isolated genes.